Ribonuclease P (RNase P) cleaves pre-tRNAs to give the mature 5' termini. RNase P holoenzymes from Bacteria, Archaea and Eucarya have been shown to contain essential RNA subunits of similar size and structure, although there are pronounced differences in the size, number and contribution of the protein components. RNase P is unusual among the known nucleolytic RNAs in that a single enzyme recognizes a large number of substrates through tertiary contacts, rather than Watson-Crick base pairing. The purpose of the proposed research is to investigate how Saccharomyces cerevisiae nuclear RNase P recognizes and cleaves its substrates and how this holoenzyme interacts with the nuclear processing pathway for tRNA primary transcripts. In the previous period, this laboratory proposed secondary and tertiary structure models for the yeast nuclear RNase P RNA. The function of highly conserved sequences and structures in the RNA models were tested by directed mutagenesis and characterization of the resulting holoenzyme assembled in vivo. To aid in biochemical characterization, we purified the nuclear RNase and identified a complex subunit structure consisting of nine proteins tightly associated with the RNA subunit. All subunits are shown to be essential for life and for RNase P function in vivo, and eight of the nine protein subunits are also associated with RNase MRP, a related endonuclease involved in -re-ribosomal RNA processing in nucleoli. This close relationship to RNase MRP caused us to examine whether RNase P might also be nucleolar, leading to the discovery that much of the pre-tRNA processing pathway, including RNase P, is located in the nucleolus. Future work will proceed from the hypothesis that a limited number of the protein subunits of RNase P participate directly with the RNA subunit in pre-tRNA cleavage, while the majority of the enzyme protein content confers functions required in the complex nuclear environment. In this proposal, several specific questions will be addressed concerning the structure and function of yeast nuclear RNase P. [1] How are RNase P substrates recognized by the RNA and protein components of this complex holoenzyme? [2] How are the subunits of the holoenzyme fit together to achieve this function? [3] What are the other roles of the protein subunits in physiological function of the holoenzyme within the nuclear tRNA processing pathway?